Human rhinovirus (RV) is the predominant etiological agent of the common cold and the leading cause of human infectious disease. RV is more than a nuisance. It is a major cause of community-acquired pneumonia in children and adults in the United States and plays a major role in triggering exacerbations of both asthma and chronic obstructive pulmonary disease, resulting in billions of dollars in medical costs every year in these high-risk populations. Decades ago, researchers identified inactivated RV as a protective vaccine, defined virus-neutralizing antibodies (nAb) as a correlate of protection against colds, and estimated duration of RV immunity using monovalent inactivated RV. However, co-circulation of many RV types discouraged further vaccine development. A 10-valent inactivated RV vaccine was tested in people in the 1970s, but only one-third of the vaccine strains induced measurable nAb, reinforcing pessimism for RV vaccine development. However, the average amount of each input RSV type antigen in this early 10-valent vaccine was low. The Moore laboratory at Emory recently published that serum nAb against many RV types can be induced by polyvalent inactivated RV adjuvanted with alum by simply increasing the amount of each RV antigen in the vaccine. Using formulations up to 25-valent in mice and 50-valent in non-human primates, inactivated RV vaccine immunogenicity was related to sufficient quantity of input antigens. Serum nAb to polyvalent inactivated RV were type-specific, necessitating a high number of valences (RV types). These proof-of-principle data point to a tractable vaccine approach for RV vaccine product development. Highly polyvalent vaccine development will require overcoming challenges in chemistry, manufacturing, and controls (CMC), such as identifying and ensuring potency and consistency of highly complex vaccine mixtures. There are three RV species, A, B, and C, with 83, 32, and 55 types, respectively. There is no evidence of RV antigenic drift. Meissa Vaccines, Inc and Emory University will initially focus on the most prevalent RV types, the 83 types within species A. In order to balance manageable CMC research with product development, our initial goal is to generate a 28-valent recombinant RV A vaccine. A 28-valent RV A vaccine process and control can later be either scaled up and adapted to higher valency or, alternatively, be developed as a series of three 28-valent vaccines, to cover all 83 RV A serotypes. In Aim 1, we will generate 28 RV A infectious clone constructs harboring the capsid proteins of types A10, A12, A15, A16, A19, A28, A29, A31, A32, A33, A34, A38, A40, A41, A49, A53, A55, A56, A58, A59, A60, A66, A68, A75, A78, A80, A85, or A96 and evaluate their ability to produce infectious virus in a cell line suitable for cGMP manufacturing. In Aim 2, we will develop a proteomics-based assay to identify and quantify unique peptides corresponding to each of the 28 types in the 28-valent vaccine composition. This assay will be advanced for measuring potency of a highly complex vaccine mixture and be assessed for ability to be qualified for releasing lots.